Prospects for lentiviral vector mediated prostaglandin F synthase gene delivery in monkey eyes in vivo

Abstract

Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.

Keywords: Glaucoma; gene therapy; lentivirus; prostaglandin synthase; trabecular meshwork.

Figure 1

Schematic diagram of the FIV vector maps for control and BOVPGFSmyc vectors. The BOVPGFS cDNA in the pUCPF1 vector was obtained from K. Watanabe (Kyoto, Japan)(53) and PCR amplified with primers that added a 5′ XbaI restriction site and replaced the 3′ stop codon with a myc epitope tag, stop codon, and EcoRI restriction site. The PCR amplicon was then gel purified and subcloned into the MCS of pZero using the XbaI and EcoRI restriction sites. Once the myc-tagged BOVPGFS construct sequence was verified, the construct was subcloned into the FIV expression vector FIV.BOVPGFS-myc.GFP using the XbaI and EcoRI restriction sites down stream of the CMV promoter. The packaging vector (pFIV-34N) contains the structural, regulatory, and replication genes that code for the proteins required for packaging the vector genome. The pVSV-G vector expresses the envelope glycoprotein of the vesicular stomatitis virus (VSV, replacing the lentiviral env gene). Structural features are indicated. LTR, Long Terminal Repeat; CMV promoter, Human Cytomegalovirus major immediate early gene promoter; MCS, multi-cloning site; copGFP, copepod GFP gene: WPRE, Woodchuck post-transcriptional regulatory element; SV40 Poly A, SV40 virus polyadenylation signal; BOVPGFS, bovine prostaglandin F synthase gene: EF-1, Eukaryotic Initiation Factor-1 promoter. Vectors were packaged according to the supplier’s instructions.

Figure 2

Schedule of procedures for individual animals. Six procedures are shown for each animal. 0) FIV injections; 1) IOP/SLE - intraocular pressure and slit lamp examination (biomicroscopy); 2) GFP photos; 3) AHF - aqueous humor formation determination by fluorophotometry; 4) OF - outflow facility determination; 5) AC taps - anterior chamber aqueous humor sampling.

Figure 3

IOP difference compared to baseline in all monkeys after vector injection. IOP after correction for baseline was significantly decreased in all FIV.BOVPGFS-myc.GFP injected eyes compared to contralateral control FIV.GFP injected eyes by ANOVA analysis for the following time periods: for all data until euthanasia (black bars), p=0.012; during the period 0-5 months (red bars), p=0.003. After 5 months (white bars) until euthanasia, the IOP was not significantly different between the eyes.

Figure 4

Detection of GFP expression in vivo after FIV.BOVPGFS-myc.GFP or FIV.GFP transduction. GFP expression was monitored serially and noninvasively as previously described (36). Briefly, after installation of 0.5% proparacaine hydrochloride, a Swan-Jacob gonioscopy lens was placed on the corneal surface of anesthetized, supine-positioned monkeys to view the iridocorneal angle and trabecular meshwork tissue. A customized Nikon microscope (Nikon Instruments) with a 12-bit, monochromatic, cooled- CCD (charge-coupled device) camera (Retiga-2000RV; Q-Imaging, Surrey, BC, Canada) and a specific GFP filter set was also used to collect images at all time points (71). Representative images are shown for monkey K691. GFP was detected with the research microscope system until shortly before euthanasia. Top row, first three panels: there is clearly expression overlying and perhaps within the ciliary muscle. Expression in the trabecular meshwork is more visible, but the images do not prove that ciliary muscle expression is negligible compared to trabecular meshwork since the depth of expression (outward from the inner aspect) could not be assessed in vivo. Presumably, deeper expression is masked by pigment in the ciliary muscle. Far right panels are from a different monkey and are shown for orientation purposes only. Little or no GFP expression was present in the 626-day post-1st injection images (both of these panels are the same magnification). The text at the bottom of figure indicates the number of days post (d p) 1^st, 2^nd, and 3^rd injections for both eyes.

Figure 5

Detailed structure of the FIV-based FIV.BOVPGFS-myc.GFP and pGINSIN vectors. hCMVp, Human Cytomegalovirus Immediate Early Promoter; 5′ LTR, 5′ Long Terminal Repeat; gag, the 5′ 230 nucleotides of the gag gene; RRE, Rev Response Element; central polypurine tract important for nuclear translocation and integration of the vector genome; PGF synthase, the bovine prostaglandin F2α synthase gene; copGFP, copepod Green Fluorescent Protein; WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element which increases mRNA stability; 3′ΔLTR, the 3′ Long Terminal Repeat containing a 167 nucleotide deletion in the U3 region important for self-inactivation, SV40 Poly-A, SV40 virus polyadenylation signal; eGFP, enhanced Green Fluorescent Protein, IRES, internal ribosome entry site that allows for internal initiation of translation of the eGFP/Neo mRNA; neo, neomycin resistance gene for selection of transduced cells. The structure of pGINSIN is from Saenz et al. (72).